Over the last 15 years on this grant, we have established that biologically active, CDR grafted humanized IgG, novel radioconjugates and toxin conjugates, many based on the anti-CD33 Ig HuM195, can be constructed, characterized, and translated into successful clinical trials in patients with AML. Most recently, we described a novel class of radio-immuno-pharmaceutical, a targetable atomic nanogenerator, with exceptional potency that has entered human clinical phase I use and that already shows anti-leukemia activity at the lowest doses. The long-term goals of the program have been to understand and improve alpha particle immunotherapy. We are now exploring the biology and radiobiology of the tumor and its environment, especially its vasculature, to better understand how to make more effective alpha-emitting agents. In the current proposal, we plan to build on this scientific foundation in targeted radioimmunotherapeutics: (1) to answer several new questions that have emerged out of our previous work targeting vasculature; (2) to explain the mechanisms of action and resistance to antibody targeted alpha-particle irradiation. Throughout this work, as before, we aim to ultimately provide therapeutic agents and concepts for their appropriate use and direct application to human clinical trials. The potency and short range of alphas makes them appealing agents for selective killing of tumor neovasculature. Initial studies showed that we could deplete and normalize residual tumor vessels in animal models with effective anti-cancer activity. In Aim 1, we plan to explore how to best utilize this finding to optimize combining alpha immunotherapy to neovasculature with agents directed to the tumor cells themselves. We also explore mechanisms by determining how the properties of the subsequent anti-tumor agents affect kinetics and tumor therapy outcomes following vessel targeting. In Aim 2, we will examine potential mechanisms of cellular resistance to alpha particle damage including: a) DNA damage sensors and transducers or apoptosis mediators or cell cycle checkpoints that distinguish alpha-resistant from sensitive cells or affect alpha sensitivity; b) DNA repair or damage mitigation mechanisms. Understanding this critical biology should allow better strategies to avoid normal cell killing and tissue damage, while enhancing tumor cell kill.